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Microbes and Infection, 2, 2000, 1724

2000 ditions scientifiques et mdicales Elsevier SAS. All rights reserved

Clinical signs, reproduction of attaching/


effacing lesions, and enterocyte invasion after oral
inoculation of an O118 enterohaemorrhagic
Escherichia coli in neonatal calves
Philippe Stordeura*, Bernard Chinaa, Gerard Charlierb, Stefan Roelsb, Jacques Mainila
a
Laboratory of Bacteriology, Faculty of Veterinary Medicine, University of Lige, Sart Tilman B43a, B-4000 Lige, Belgium
Section of Pathology, Department of Biocontrol, Centre for Research in Veterinary Science and Agrochemistry, Groeselenberg 99, B-1180 Brussels, Belgium

(Received 8 July 1999; accepted 30 September 1999)

ABSTRACT Attaching and effacing (AE) lesions are produced among others by enteropathogenic
Escherichia coli and enterohaemorrhagic E. coli (EHEC), which differs from the former by the production
of cytotoxins active on various cell cultures, the verocytotoxins, or shigacytotoxins. EHEC are
associated with diarrhoea and dysentery in humans and in ruminants, mainly calves from two to eight
weeks of age. Clinical signs and/or lesions have been reproduced experimentally with EHEC strains
belonging to serotypes O5:K4/Nm, O26:K-:H11, O111:Nm, and O157:H7 which are isolated from
cattle and/or humans. The purpose of this work was to develop an experimental model of infection in
newborn calves with a bovine EHEC strain isolated from a calf which of died of diarrhoea, and
belonging to the O118:H16 serotype, which is also common to both cattle and humans. The bovine
O118:H16 EHEC strain was able to colonize the gut of three newborn calves, and to induce diarrhoea
twenty-four hours after challenge and to produce AE lesions in the small and/or large intestines. AE
lesions were detected microscopically and ultrastructurally in the small intestine of one calf and in the
whole intestinal track of two calves. Internalization of bacteria and also of pedestal-bacteria complex
inside of the enterocyte was observed in two of the three calves. The significance of this stage is
unknown but may be related to the invasion of the calf by the bacteria. The challenge strain was
isolated from the mesenteric lymph nodes of the same two calves but not from other organs or from
heart blood. No blood was observed in the faeces of any of the three calves, nor were any lesions in the
internal organs, which may have been related to the production of a verotoxin whose role is still
unknown in cattle. 2000 ditions scientifiques et mdicales Elsevier SAS
enterohaemorrhagic E.coli (EHEC) / calf / attaching and effacing lesions (AE) / diarrhoea / invasion

1. Introduction
Since 1984 [2], attaching and effacing Escherichia coli
is recognised as a cause of diarrhoea and dysentery in
young calves, mainly from two to eight weeks of age [5,
16]. Attaching and effacing was the term first used by
Moon and collaborators [22] to describe an intestinal
lesion (AE lesion) caused by specific strains of E. coli:
effacing because of the localized disappearance of the
brush border microvilli; attaching because of the inti-

mate attachment of the bacteria to the exposed cytoplasmic membrane of the enterocyte.
AE lesions are caused by enteropathogenic E. coli
(EPEC), Citrobacter rodentium, Hafnia alveii, and enterohaemorrhagic E. coli (EHEC), which differ essentially from
the EPEC by the production of cytotoxins active on various
epithelial cells (Hela and Hep-2) in culture, the verocytotoxins (VT1 and VT2 families), also named shigacytotoxins
(Stx1 and Stx2 families) [19, 21, 24]. EPEC causes diarrhoea in various animal species and in humans, whereas
EHEC is associated with diarrhoea and dysentery in rumi-

* Correspondence and reprints


Microbes and Infection
2000, 17-24

17

Original article

nants and in humans. In the latter, EHEC also causes the


haemolytic-uraemic syndrome (HUS) [4, 16, 19, 24, 25].
The pathogenesis of EPEC/EHEC and the production of
the AE lesions have been intensively studied in vitro on
cells in culture with the human EPEC strain E2348/69, and
a four-step model has been tentatively proposed [24],
consisting of (i) initial adherence to the enterocyte
microvilli; so far fimbrial adhesins have been described
only for human EPEC [9] and rabbit EPEC [21]; (ii) type III
secretion system-mediated signal transduction into the
enterocyte resulting in increased levels of phosphorylation
and calcium, as well as in cytoskeleton rearrangements.
(In vivo, the most dramatic consequence is the effacement
of the brush border microvilli); (iii) intimate attachment to
the exposed enterocyte cytoplasmic membrane, mediated
by an outer membrane protein, intimin, with polymerisation of actin filaments underneath the zone of adherence
of the bacteria and enhancement of the cytoskeleton rearrangements; and (iv) penetration of the bacteria into the
cells. The VT/Stx toxins play no role in the development of
the AE lesions, but cause damage to the intestinal wall
vessels resulting in haemorrhages and dysentery in
humans and calves. In humans, they also cause damage to
the arterioles of the kidneys, resulting in the HUS [19, 24,
25].
The first step is mediated by genes located on a plasmid
or on the chromosome [21, 24]. Genes responsible for
steps (ii) and (iii) are grouped together on the bacterial
chromosome forming a pathogenesis island, the locus of
enterocyte effacement, or LEE [20]. The genetic determinism of the fourth step is still unknown although chromosomal mutants deficient only in cell invasion have been
described [8]. The genes coding for the VT/Sta are also
located on the chromosome, but on phages for several of
them [19].
The transposition in vivo of these in vitro models has
been realized for the first three steps with human and
rabbit EPEC by testing mutants [10, 21], but the in vivo
significance of the fourth step, the cellular invasion, is still
unknown. Bovine EPEC and EHEC also possess a LEE [12],
and AE lesions and /or clinical signs have been experimentally reproduced in young calves and lambs, with bovine
EHEC belonging to various serotypes: O5:K4:Nm, O26:K:H11, O111:Nm, and O157:H7 [1, 7, 18, 23, 29, 32].
Bovine EPEC and EHEC belonging to other serotypes have,
however, not been tested in vivo yet.
The purpose of this work was to develop an experimental model of infection in newborn calves to confirm the
pathogenicity of and follow the production of AE lesions
by a bovine EHEC strain belonging to serotype O118:H16.
This model will help to study mutants in various
pathogenicity-associated genes of bovine EHEC in the
future.

2. Material and methods


2.1 Bacterial strain

Strain 340S89 is an O118:H16 E. coli which was isolated in 1989 from a two-week-old Friesian calf which
died of diarrhoea [27]. It tests positive with the gene
18

Stordeur et al.

probes for intimin (Eae probe) and VT1, but not with the
gene probe for VT2 [17] and is able to reproduce AE
lesions in rabbits [4]. This strain is sensitive to kanamycin,
gentamycin, tetracycline, chloramphenicol, and nalidixic
acid, but resistant to streptomycin and tellurite.
2.2 Calf infection

Four naturally born calves were isolated immediately


after birth in a box which had been washed and desinfected (Atlantol*, Ecosa, Ghent, Belgium). They received
300 millilitres of colostrum which was negative by ELISA
and agglutination against strain 340S89. At six hours of
age, three of them were challenged orally with 109 to 1010
CFU of strain 340S89 suspended in sterile saline and the
fourth one with saline only. Strain 340S89 was grown on
Meat extract Agar slants (Oxod, Gent, Belgium) for six
hours aerobically at 37 C. The bacteria were resuspended
in sterile saline and the optical density at 620 nm was
adjusted to obtain a bacterial concentration between 109
to 1010 per 200 mL. The exact concentration was calculated after inoculation of tenfold dilutions of the suspension onto Gassner agar plates (Belgolabo, Overijse, Belgium). This medium was used to select enteric bacteria.
Moreover, it provided the opportunity to make a differentiate between coliforms thanks to his lactose activity [11].
The calves were subsequently fed twice a day with two
liters of UHT whole milk. Clinical investigations and faecal sampling were performed every four hours. Faecal
samples were tested by ELISA (trousse ELISA digestive,
BioX, Brussels, Belgium) for the presence of K99 enterotoxigenic E. coli, rotavirus, coronavirus, and Crytosporidium sp. The excretion of the challenge strain was followed by bacteriological examination and by PCR.
2.3. Necropsy

The calves were euthanised between 44 and 64 h p.i.


(p.i.) by intravenous administration of sodium pentobarbital. Necropsy was performed on a general routine basis
at first and subsequently focused on the abdominal cavity.
Samples were taken aseptically from various intestinal
segments (duodenum, jejunum, ileum, caecum, spiral
colon, descending colon, and rectum) and from internal
organs (mesenteric lymph nodes, liver, spleen lungs, kidneys, heart blood, and brain) for bacteriology, histopathology, and electron microscopy.
2.4. Bacteriology

The faecal samples were collected in sterile containers.


The postmortem faecal samples were taken aseptically
(the sections of the gut had been sterilized using burning
steel) with a Pasteur pipette. The samples were then
diluted tenfold. Dilutions 104 to 106 were inoculated
onto Gassner agar and Gassner agar supplemented with
streptomycin sulphate (Sigma Aldrich, Steinheim, Germany,100 g per mL) and tellurite (Sigma Aldrich, Steinheim, Germany,104 M) plates with a spiral plater system
(LED Techno, Hechtel-Eksel, Holland), and incubated
overnight aerobically at 37 C. After one night of incubation, a calculation of the number of bacteria on the plate
was realized and this result was transformed in CFU/g of
intestinal content by a formula.
Microbes and Infection
2000, 17-24

Enterohaemorrhagic E. coli infection in calves

Original article

Figure 1. Faecal excretion of strain 340S89 (Log CFU/ mL of faeces) and appearance of diarrhoea as a function of p.i. time. The calves were
euthanazied at 56 hours p.i. (calf 1), 64 hours p.i. (calf 2), and 44 hours p.i. (calf 3), respectively.

Each lymph node was investigated before intestines


were opened. The content of this one (taken with a Pasteur
pipette after sterilization of the surface using burning steel)
was seeded directly onto the selective medium (Gassner
agar plates supplemented with streptomycin sulfate and
tellurite).
For each faecal sample, five colonies grown on the
streptomycin-tellurite Gassner agar plates at 106 concentration were compared with strain 340S89 by tube agglutination with the O118 immunserum, by determination of
the antibiotic sensitivity, by PCR, and by pulsed-field gel
electrophoresis (PFGE). In addition, colonies of one plate
for each sample were tested by colony hybridization with
the probes for intimin and VT1 after direct transfer onto
filters.
2.5. Tube agglutination and antibiotic sensitivity

The tube agglutination test with an O118 immune


serum was performed following standard procedure [30].
The antibiotic sensitivity was performed on MuellerHinton II agar (Becton Dickinson Benelux, Erembodegem,
Belgium) with the following antibiotic discs according to
the manufacturers instructions: tetracyclin (30 g),
chloramphenicol (30 g), streptomycin (10 g), gentamycin (10 g), kanamycin (30 g), nalidixic acid (30 g),
ampicillin (10 g), and rifampicin (5 g).
2.6. Colony hybridization, PCR, and PFGE

The colony hybridization assay was performed with


gene probes for the intimin (Eae probe) and for VT1(VT
probe) as described by Mainil and collaborators [17]. The
probes were labelled with [-32P] dCTP by random priming using the dCTP-labelling beads (Ready to go, Pharmacia, Uppsala, Sweden). A PCR specific for the eae gene
was performed on the faecal samples and on the internal
organs with a positive culture, and a multiplex PCR for the
intimin and verotoxin genes was performed on the faecal
samples as described by China and collaborators [3].
PGFE was performed on a CHEF mapper apparatus (BioMicrobes and Infection
2000, 17-24

rad laboratories, Hercules, California, USA) using the following parameters: voltage of 6V/cm, migration time of
21 h, switch time of 2 to 20 s, angle of 120, ramping
factor of 1 379. The plugs were prepared using the Genpath group 2 reagent kit (Bio-Rad) and digested for five
hours with Xba1 (Gibco, Paisley, Scotland, UK).
2.7. Histopathology and electron microscopy

Tissues were fixed in a phosphate-buffered


formaldhehyde/glyceraldehyde solution (4%/1%), processed routinely, and paraffin embedded, and sections of
5 m were obtained. Tissue sections were stained with
hematoxylin-eosin and examined for presence of bacteria
and inflammatory lesions. For transmission and scanning
electron microscopy, the samples were fixed in the same
solution. Samples for transmission electron microscopy
were embedded in Epon/Spurr (50/50) and ultrafine sections were examined in a Philips 208S electron microscope. Samples for scanning electron microscopy were
critical point dried, gold sputtered and examined in a
Philips 501 electron microscope.

3. Results
3.1 Clinical signs

The three calves inoculated with strain 340S89 had


acute serous non-bloody diarrhoea beginning 24 h p.i.,
and which lasted for 12 h in two of them (calves 1 and 3)
and until the euthanasia in calf 2 (figure 1). A mild hyperthermia was also noted (39.6 to 39.9 C), but only calf 2
had general clinical signs such as inappetence, prostration, and congestion of mucosae. The control calf had
neither diarrhoea nor general clinical signs.
3.2. Faecal excretion

The challenge E. coli strain 340S89 was detected in the


faecal samples of the three challenged calves from
between 8 to 20 h p.i. until euthanasia (figure 1), after
19

Original article

Stordeur et al.

Table I. Concentration of the strain 340S89 (CFU/mL)


in different segments of the gut.
Duodenum
Jejunum
Ileum
Caecum
Spiral colon
Descending colon

Calf 1

Calf 2

Calf 3

Control

0
0.2 107
1.2 108
1 107
1.6 108
3 107

0
1.7 109
2.6 109
1.3 109
2.6 109
0.2 107

0
0
1.4 108
0.9 109
7 108
0.2 107

0
0
0
0
0
0

growth on streptomycin-tellurite gassner agar plates and


identification by tube agglutination with an O118 immune
serum, by antibiotic sensitivity determination, by DNA
colony hybridization with the Eae and VT1 probes and by
PFGE as described in the Material and methods section.
The peak of faecal excretion was observed between 20 to
24 h p.i.. Strain 340S89 was excreted by most calves at
concentration between 105 and 1010 CFU/g of faeces
(figure 1) and represented 65 to 100% of the total coliform
population. In the samples, concentrations of 1011 to 1012
CFU/g of faeces were obtained (figure 1) but most probably result from a technical problem.
The PCR results for the eae gene on the faecal samples
were positive from between 16 to 20 h p.i. until euthanasia.
The control calf never excreted the challenge strain nor
an eae PCR-positive E coli. All four calves remained
negative for presence of coronavirus, Cryptosporidium
and K99-enterotoxigenic E. coli. Only calf 2 was found
positive for excretion of rotavirus as early as 24 h p.i..
3.3. Necropsy

The three calves presented lesions of acute serous


enteritis and/or colitis: lesions were localized on the large
intestine (calf 1) or extended throughout the whole intestinal tract (calves 2 and 3). No lesions were found in the
internal organs with the exception of the mesenteric
lymph nodes which were enlarged. The control calf
showed no lesion at all.
3.4. Postmortem bacteriology

E. coli strain 340S89 was detected and identified, as in


the faecal samples, in various intestinal segments of the
three challenged calves (table I) and from the mesenteric
lymph nodes of calves 2 and 3. Strain 340S89 was present
at concentrations varying between 106 to 109 CFU/g of
intestinal content and represented 60 to 85% of the total
coliform population. Strain 340S89 was not detected in
the control calf either from the intestine or from the
intestinal organs. PCR performed on the mesenteric lymph
nodes of calves 2 and 3 were positive, and negative on the
mesenteric lymph nodes of the control calf.
3.5. Histopathology and electron microscopy

An important inflammatory reaction with neutrophil


and lymphocyte infiltration (figure 2A) was observed in
the wall of most segments of the intestine of the three
challenged calves (table II). Adherent bacteria were vis20

ible in the large intestine of calf 1 and in the small and


large intestine of calves 2 and 3. When adherent bacteria
were present, the brush border was no longer visible
(figure 2A). The Peyers patches and the mesenteric lymph
nodes showed low to intensive degrees of activation
(table II). No lesion was observed in the internal organs
including the brain. The control calf showed no lesion
other than vascular congestion in all organs and some
degree of cellular infiltration in parts of the intestine
(table II).
Scanning and electron microscopy confirmed the presence of closely adherent bacteria and of localized effacement of the enterocyte microvilli where the bacteria
attached (figure 2B, 2C). Pedestal and actin accumulation
under the zone of adherence of the bacteria were also
present (figure 2C). Moreover, internalization into the
enterocytes of bacteria and of bacteria-pedestal complexes were evident in calf 2 (figure 2D). None of these
lesions were observed in the control calf.

4. Discussion
In this work an O118:H16 EHEC strain was able not
only to colonize the gut of three newborn calves, but also
to induce diarrhoea 24 h after challenge and to produce
AE lesions in the small and/or large intestines. The
appearence of diarrhoea coincides with the peak of faecal
excretion of the challenge strain, but lasted only for 12 h in
two calves (figure 1). Faecal excretion of the challenge
strain was detected earlier by growth on a selective agar
containing streptomycin and tellurite than by PCR on
faecal samples.
As no other classical diarrhoeagenic infectious agents
were isolated from these two calves and as the control calf
did not develop diarrhoea [7], the O118:H16 EHEC strain
can be considered the cause of diarrhoea. It may be
argued that challenge with any bacteria would cause the
appearance of diarrhoea. Arguments in favour of a role of
the O118:H16 EHEC strain are: (i) challenge with a nontoxigenic F17-producing E. coli strain resulted in the
absence of any clinical signs (Van Bost and Mainil, unpublished data); (ii) the colonization of the gut was still high as
attested by the faecal excretion even after the disappearance of the diarrhoea (figure 1). The direct cause of diarrhoea by EPEC/EHEC strains is believed not to be the AE
lesions, however extended they can be, and is actually still
unknown, although an active mechanism based on ion
exchange and mediator perturbation is under consideration [24].
The severity and the length of the diarrhoea was thus
not related to the degree of intestinal colonization, nor to
the degree of extension of the macroscopic (acute serous
enteritis) and microscopic (AE) lesions (small intestine for
calf 1, small and large intestine for calves 2 and 3; table II).
Other factors must thus be considered: the presence of
other diarrhoeagenic agents, such as a rotavirus in calf 2;
the age of the animal and its genetic background; loss of
some bacterial virulence genes during storage. Coexistence between EPEC/EHEC strains and other diarrhoeagenic agents has already been noticed in field studies and
Microbes and Infection
2000, 17-24

Enterohaemorrhagic E. coli infection in calves

Original article

A
B

C
Figure 2. Histological and ultrastructural lesions present in the
gut. A. Light micrograph of descending colon of calf 3. Bacteria
attached to the top of the enterocytes (1), presence of lymphocyte and
polymorphonuclear leucocytes (2), and area without bacteria (3). The
bar corresponds to 25 m. B. Scanning electron microscopy of spiral
colon of calf 2: microvilli effacement is clearly visible (4) as are
bacteria attached to the top of the microvilli (5). The bar corresponds
to 10m. C. Transmission electron microscopy of the spiral colon of
calf 2: bacteria adhere to the enterocyte cytoplasmic membrane (6);
pedestal and actin accumulation (7) are clearly visible. The bar
corresponds to1m. D. Transmission electron microscopy of the
ileum of calf 2: bacteria internalization (8) and a pedestal-bacteria
complex in a phagosome (9) are visible inside of the enterocyte.
D
may be related to the severity and persistance of diarrhoea
in farms [5]. The age of the animal is also important: with
strains of serotypes O157:H7 and O26:H11 clinical signs
could not be reproduced in calves aged one to two weeks
but could be reproduced well in newborn calves [1, 6, 7,
18]. The opposite situation may exist for strains belonging
to other serotypes, mimicking the situation of EPEC in
rabbits [21, 26].
VT toxins are thought to cause the appearance of
bloody diarrhoea at least in man [24], but the situation
may be different in calves. No blood was indeed observed
in the faeces of any of the three calves, nor were there any
Microbes and Infection
2000, 17-24

lesions in the internal organs which may have been related


to the production of a verotoxin, in the brain as in piglets
suffering from oedema disease [19] or in the kidneys as in
humans suffering from HUS [24]. The role of VT1 produced by most cattle EHEC [17, 19] is actually still
unknown. Challenge experiments with the wild-type
strain and isogenic mutants in the genes coding for the
verotoxins may bring an answer to this question.
An often overlooked property of at least some EPEC/
EHEC strains is their capacity to invade cells in vitro [8]
and in vivo [28]. Internalization of bacteria and also of
bacteria-pedestal complex inside the enterocyte was
21

Original article

Stordeur et al.

Table II. Histological and ultrastructural lesions and observations.


Calf 1

Calf 2

Calf 3

Control

PMNc, Ld
+
b

PMN

L, Me
+

Ef, M

L, M, PMN
+
+

L, M, PMN

L, M, PMN

+
+
+

Congestion

Infl. cells.
Abcess
Peyers patches
Villosity atrophy
AE lesions
Internalization

PMN, L
+
+

PMN, L, M

+
+
+
+

PMN, M, L

+
+
+
+

E, M

Caecum

Congestion
Infl. cells.
Abcess
Villosity atrophy
AE lesions
Internalization

+
PMN

+
PMN

+
+
+

+
PMN, L, E

+
+

PMN

Spiral colon

Congestion
Infl. cells.
Abcess
Villosity atrophy
AE lesions
Internalization

+
L

+
PMN

+
+
+

L, PMN, E

Descending colon

Congestion
Infl. cells.
Abcess
Villosity atrophy
AE lesions
Internalization

+
PMN, L

+
PMN, M

+
+

L, PMN

Lymph node

Congestion
Bacteria
Activity
Infl. cells.

Intensive

Intensive
PMN

Intensive
PMN, N

Restricted
PMN

Brain

Congestion
Infl. cells.
Bacteria

Duodenum

Congestion
Infl. Cellsg.

Villosity atrophy
Abcess
AE lesions
Internalization
Jejunum

Congestion
Infl. cells.

Abcess
Peyer spatches
Villosity atrophy
AE lesions
Internalization

Ileum

presence; b absence; c polymorphonuclear leukocytes; d lymphocytes; e macrophages; f eosinophiles; g inflammatory cells.

observed in two of the three calves (table II). It is also


worth noting that strain 340S89, and several other EHEC
and EPEC bovine strains, give images of internalization
into the enterocytes when tested in the rabbit ligated
22

intestinal loop assay [4, 12]. The significance of this stage


is unknown but may be related to the invasion of the calf
by the bacteria. Indeed, the challenge strain was isolated
from the mesenteric lymph nodes of the same two calves.
Microbes and Infection
2000, 17-24

Enterohaemorrhagic E. coli infection in calves

However the challenge strain was never detected in other


organs or in heart blood.
In summary, bovine EHEC strain 340S89 belonging to
serotype O118:H16 and isolated from a calf with diarrhoea, was able to induce diarrhoea and to produce AE
lesion in newborn calves. Presence of other infectious
diarrhoeagenic agents can influence the severity and the
length of the diarrhoea. This model can be used for further
challenge experiments to study the role of the different
genes of the LEE and of VT1 in the pathogenesis of bovine
EHEC strains and to study the host specificity of EHEC
strains belonging to the same serotype and isolated from
both cattle and humans.

Acknowledgments
We thank Dr C. Manteca from Provincial Laboratory of
Loncin (Lige, Belgium) for performing the Trousse ELISA
digestive assay. Philippe Stordeur was supported by a
studentship from the Ministry of Middle Classes and Agriculture. This work was also financially supported by the
Ministry of Middle Classes and Agriculture (convention
5740 A).

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